Study On The Function And Mechanism Of ZGRF1 In The Occurrence And Progression Of Non-small Cell Lung Cancer

Project background and purposeAt present,lung cancer is the malignant tumor with the highest morbidity and mortality in the world and China.Of these,80-85%are NON-small cell lung cancer(NSCLC).In the United States,55%of patients with NON-small cell lung cancer have distant metastasis at the time of first diagnosis.The 5-year survival rate of stage IVA and stage IVB NON-small cell lung cancer is approximately 10%and less than 1%,respectively.The traditional treatment for advanced lung cancer has been chemotherapy,with limited survival benefits.In recent years,with the in-depth research on the molecular biology of lung cancer and the development of targeted drugs and immune checkpoint inhibitors,the diagnosis and treatment of lung cancer has been significantly improved,but limited to the current drug indications,drug resistance and serious adverse reactions,only a few patients benefit.Therefore,the search for new biomarkers and drug targets is the focus of lung cancer research.Human ZGRF1 is one of many helicases that encode proteins containing GRF zinc fingers and transmembrane domains.GRF zinc fingers are present in many DNA-binding proteins,and selective splicing results in multiple transcriptional variants encoding different isoforms.It plays an important role in homologous recombination repair(HR)and DNA cross-linking(ICL)repair.Studies have confirmed that ZGRF1 knockout cells are sensitive to MMC,topoisomerase I(TOP1)inhibitor and camptoctin(CPT).In addition,ZGRF1 has a positive up-regulation effect on phenotype and biological function of breast cancer stem cells in vitro.High expression of ZGRF1 in triple negative breast cancer is associated with poor prognosis.However,its specific role and mechanism in the occurrence anddevelopment of NON-small cell lung cancer has not been reported,which is worthy of further research.·Bioassay and immunohistochemical detection of lung cancer tissue chip showed that ZGRF1 expression level in lung cancer tissue was significantly higher than that in paracancer tissue,and its expression level was significantly correlated with clinical stage and T stage.High expression of ZGRF1 was associated with poor prognosis.It was confirmed in vitro that ZGRF1 could significantly promote the proliferation and migration of cancer cells and inhibit apoptosis.Knockdown of ZGRF1 significantly inhibited the growth of subcutaneous transplanted tumor in nude mice.The ZGRF1 interacting protein was screened by IP-MS proteomics and verified by co-IP that EIF4A3 was ZGRF1 interacting protein.Signaling pathway protein detection and pathway protein activator assay of ZGRF1 knockdown group and control group cells,nude mouse transplanted tumor and human NON-small cell lung cancer tissues confirmed that ZGRF1 may be involved in the regulation of PI3K/AKT signaling pathway by binding EIF4A3 protein.In this study,in vitro and in vivo experiments were conducted to confirm the effects of ZGRF1 on the biological behavior of NON-small cell lung cancer,and proteomics techniques were used to find the signaling pathways involved in the regulation of interacting proteins,aiming to clarify the specific mechanism of ZGRF1.To provide the corresponding theoretical basis for the transformation of clinical achievements.[Objective]To investigate ZGRF1 expression and explore the correlation between ZGRF1 expression and pathological features and overall survival in non-small cell lung cancer.[Methods]1.Bioinformatics methods were used to analyze the expression and prognostic value of ZGRF1 in lung squamous cell carcinoma and lung adenocarcinoma.We used the GDC download tool officially provided by TCGA to select the paired sample data of RNAseq for analysis.Differential analysis of ZGRF1 gene expression profile in lung squamous cell carcinoma and lung adenocarcinoma using TCGA database DESeq2 method,Chisq Test was used for statistical analysis of the correlation between ZGRF1 gene expression level and clinical stage and other pathological features.Prognostic analysis based on clinical information of TCGA-LUAD and TCGA-LUSC samples,kaplan-Meier Plotter was used to analyze the prognostic effect of ZGRF1 expression in lung cancer patients.2.The expression of ZGRF1 was detected by immunohistochemical staining in lung cancer tissue sections.The difference analysis of ZGRF1 expression between tissue chip carcinoma and adjacent tissues was carried out by Chi-square test.Mann-Whitney U Test and Spearman rank correlation analysis were used to analyze the correlation between ZGRF1 expression level in cancer tissues and clinical data.Kaplan-meier method(log-rank test statistics)was used for survival analysis.[Results]1.Differential expression of ZGRF1 gene1)Gene expression profile analysis results of TCGA database:ZGRF1 gene was differentially expressed in lung squamous cell carcinoma(log2FC=1.4419 p=7.8835E-42)and lung adenocarcinoma(log2FC=1.1178 p=2.9534E-19),and significantly higher in tumor tissues than in adjacent tissues.2)Tissue chip detection results:The expression of ZGRF1 in tumor tissues of 84 NSCLC patients was significantly higher than that in adjacent tissues.(p<0.001).2.Correlation analysis between ZGRF1 gene expression level and clinicopathological indicators1)The expression of ZGRF1 in lung squamous cell carcinoma of TCGA patients was significantly correlated with clinicopathological indicators such as clinical stage(p=4.0936E-07)and T stage(p=1.2581E-05).The expression level of ZGRF1 in lungadenocarcinoma tissues was significantly correlated with clinical stage(p=2.097E-21),T stage(p=2.171E-16)and N stage(p=3.233E-15)2)Tissue microarray detection results:The expression level of ZGRF1 in cancer tissues was significantly correlated with clinical stage(p=0.001**),T stage(p=0.002**)and M stage(p=0.019*).3.Results of tissue chip survival analysis:Patients with low expression of ZGRF1 had longer survival(p=0.030).[Conclusion]1.ZGRF1 was differentially expressed in NSCLC and significantly up-regulated in tumor tissues.2.ZGRF1 expression was significantly correlated with Stage,Pathologic T in lung cancer.3.High expression of ZGRF1 gene is associated with poor prognosis[Objective]To explore the role of ZGRF1 in the occurrence and progression of non-small cell lung cancer by gene knock-down in H1299 cell lines and A549 cell lines.[Methods]1.Construction of ZGRF1 knock-down lentivirus and validation of knock-down efficiency.2.Effects of ZGRF1 knock-down on lung cancer cells.(1)CCK8 assay was used to detect cell proliferation.(2)Cell migration ability was detected by cell scratch test.(3)Apoptosis was detected by flow cytometry.3.Tumorigenesis experiments in nude mice were performed to verify the effect of ZGRF1 on the growth and survival of non-small cell lung cancer in vivo.[Results]1.Effects of knockdown of ZGRF1 gene on proliferation,migration and apoptosis of lung cancer cells1)compared with the control group(shCtrl),the proliferation ability of nci-h1299 and A549 cells in ZGRF1 knockdown group(shZGRF1 group)was significantly decreased after 96h(p<0.001);2)compared with the control group(shCtrl),the migration ability of nci-h1299 cells and A549 cells in shZGRF1 group was significantly decreased after 24 and 48h(p<0.001);3)Compared with the control group(shCtrl),the apoptosis rates of NCI-H1299 cells and A549 cells in shZGRF1 group were significantly increased(P<0.001).2.The tumorigenesis experiments in nude mice.(1)Compared with the control group,the tumor weight and fluorescence expression of shZGRF1 group in NCI-H1299 and A549 cell lines were significantly decreased(p<0.05).(2)Compared with the control group,the tumor growth of shZGRF1 group in NCI-H1299 cells and A549 cell lines was significantly decreased(p<0.05).[Conclusion]ZGRF1 promoted the proliferation and the migration of NSCLC cell lines and inhibited the apoptosis of NSCLC cell lines in vitro.ZGRF1 promoted the growth of NSCLC cell lines in nude mice in vivo.[Objective]The interaction proteins of ZGRF1 was screened by IP-MS and verified by CO-IP to identify the ZGRF1 interaction proteins.[Methods]1.The lentivirus vector plasmid with 3*Flag-ZGRFl overexpression was constructed and packaged as lentivirus vector to transfect NCI-H1299 cells2.IP-MS proteomics was used to detect ZGRF1 interacting proteins3.GO analysis and KEGG signal pathway enrichment analysis assisted the screening of ZGRF1 interacting proteins4.String protein interaction database query ZGRF1 protein interaction database5.Co-IP verified protein interaction[Results]EIF4A3 was identified as an interacting protein with ZGRF1 protein.[Conclusion]It was confirmed that EIF4A3 is a ZGRF1 interacting protein,and ZGRF1 may promote the proliferation and migration of lung cancer cells and inhibit the apoptosis of cancer cells by binding EIF4A3 protein[Objective]To further explore the molecular mechanisms of ZGRF1 protein binding EIF4A3 complex to regulate downstream signaling pathways.[Methods]1.The protein expressions of ZGRF1,AKT,p-Akt,ERK,p-ERK,P38,p-P38,PI3K and p-PI3K in NCI-H1299 and A549 cell lines were detected by WB respectively.2.Signal pathway agonist experiments.WB detected the protein expressions of ZGRF1,AKT and p-AK T,ERK,p-ERK,P38,p-P38,PI3K,p-PI3K in NCI-H1299 and A549 cell lines,namely empty plasmid control group(shCtrl),ZGTRF1 knock-down group(shZGRF1),empty plasmid control group(shCtrl)+AKT activator and ZGRF1 knock-down group(shZGRFl)+AKT activator.3.The protein expressions of ZGRF1,PI3K,p-PI3K,AKT and p-Akt in tumor tissues of nude mouse xenografts were detected by WB(ZGRF1 knock-down group and the control group in NCI-H1299 and A549 cell lines were totally 4 groups).4.The protein expressions of downstream signaling pathway in 4 clinical tumor tissues and paired para-cancer tissues were detected by WB.[Results]1.Downstream signaling pathway protein expression results of the four groups:1)Protein detection results of NCI-H1299 cell signaling pathway:Compared with shCtrl,protein expressions of ZGRF1,p-Akt and p-PI3K were down-regulated in shZGRF1 group,while p-P38 protein expression was up-regulated,and p-ERK protein had no significant change.2)A549 cells:Compared with shCtrl,protein expressions of ZGRF1,p-Akt,p-ERK,p-P38 and p-PI3K were down-regulated in shZGRF1 group.2.Experimental results of AKT agonist signaling pathway2.1 Experimental results of AKT activator in NCI-H1299 cells;1)Compared with shCtrl,protein expressions of p-Akt,p-P38,ZGRF1 andp-ERK were down-regulated in shCtrl+AKT activator group,while p-PI3K protein had no significant change.2)Compared with shZGRF1,the protein expressions of p-PI3K and p-P38 in shZGRF1+AKT activator group were down-regulated,and the protein expressions of p-Akt,p-ERK and ZGRF1 were up-regulated.2.2 A549 Cell AKT agonist results:1)Compared with shCtrl,the protein expressions of p-Akt and ZGRF1 in shCtrl+AKT activator group were down-regulated,the protein expressions of p-PI3K and p-ERK were up-regulated,and the protein expression of p-P38 was not significantly changed.2)Compared with shZGRF1,the expression of p-ERK protein in shZGRF1+AKT activator group was down-regulated,the expression of p-PI3K and ZGRF1 protein was up-regulated,and the expression of p-Akt and p-P38 protein was not significantly changed.3.Protein expression of downstream signaling pathway in nude mouse xenograft tumor tissues(NCI-H1299 and A549 cell lines).(1)NCI-H1299 cells transplanted tumor detection results:Compared with shCtrl group,the protein expressions of p-PI3K and ZGRF1 in shZGRF1 group was decreased,and the protein expression of p-Akt was increased.(2)A549 cells transplanted tumor detection results:Compared with shCtrl group,the protein expressions of ZGRF1,p-Akt and p-PI3K in shZGRF1 group were down-regulated.4.The protein expressions of downstream signaling pathway was detected by WB in clinical tumor tissues.The protein expressions of p-Akt,p-ERK,p-P38 and p-PI3K were positively correlated with the protein expression of ZGRF1 in adjacent tumors.[Conclusion]ZGRF1 may regulate the PI3K/AKT signaling pathway in NSCLC by binding to EIF4A3 protein,thereby affecting the proliferation,migration and apoptosis of tumor cells,and play a carcinogenic role in the occurrence and development of NSCLC and affect the prognosis of patients.Therefore,ZGRF1 is likely to be a new biomarker and potential target for lung cancer.